Project Summary This project concerns IL-4 receptor (IL-4R) reprogramming of antigen receptor (BCR) signaling in B cells. Much study of signaling in the past has focused on biochemical events triggered in nave B cells by BCR engagement alone. This has led to identification of signalosome mediators that form the ?classical? signaling pathway and play an important role in B cell activation, but, the study of nave B cells does not consider interactions among receptors that determine the ultimate outcome of stimulation. We have shown that prior exposure to IL-4 alters the nature of subsequent BCR signaling in normal and in malignant (CLL) B cells. Following IL-4 treatment, an ?alternate? pathway for BCR signaling is created that is completely signalosome-independent. The proposed study aims to identify the fundamental biochemical changes that are produced by IL-4 to establish the alternate pathway. Lyn is absolutely required for alternate pathway signaling, whereas it has been known for years that Lyn is not required for classical pathway signaling. An early event that is completely specific for alternate pathway signaling is phosphorylation of PKC? by Lyn, which occurs only when BCR triggering follows IL-4 exposure in normal and malignant B cells. Curiously, levels of Lyn and PKC? are similar in nave and IL-4- treated B cells, indicating that protein components that interact after BCR reprogramming are available in nave B cells, yet signaling via the alternate pathway does not occur in nave B cells, only in IL-4-exposed B cells. This means that something occurs after IL-4R engagement that alters the relationship of key signaling mediators in such a way that activities/interactions that did not occur previously, do so now. This provides a discrete system with which to determine molecular changes responsible for creating the alternate pathway. We will do this by examining Lyn phosphorylation of PKC?, using this as a key and unique signature of alternate pathway signaling. We will evaluate 2 principal possibilities: 1) Lyn or PKC? is changed, whereby Lyn substrate preference is altered such that PKC? is a more avid target vs PKC? efficiency as a Lyn src kinase substrate is altered, leading to Lyn-PKC? interaction and PKC? phosphorylation; or, 2) PKC? is translocated to a membrane site conducive to Lyn-directed phosphorylation leading to Lyn-PKC? interaction and PKC? phosphorylation. This proposal represents a new, molecular direction for an established line of study. The high reward in this study is a much deeper understanding of how B cells, including malignant B cells, actually become activated in the milieau of cytokines that are experienced physiologically and pathologically in vivo. The results of this study will point the way to further dissection of the mechanism responsible for alternate pathway signaling by determining whether the focus should be on changes in the nature of Lyn vs PKC?, or changes in PKC? location and translocation. This will reveal further insights into normal B cell activation as well as signaling in CLL cells.